DEtail-seq profiled meiotic DSBs in Homo sapiens
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ABSTRACT:
PROVIDER: PRJNA645446 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12672121_1.fastq.gz | Fastqsanger.gz | |||
| SRR12672121_2.fastq.gz | Fastqsanger.gz | |||
| SRR12672122_1.fastq.gz | Fastqsanger.gz | |||
| SRR12672122_2.fastq.gz | Fastqsanger.gz |
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Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are non-randomly distributed across the genome. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to map the distribution of meiotic DSBs in an SK1 x S88C F1 hybrid strain of Saccharomyces cerevisiae.
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Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are non-randomly distributed across the genome. Here, we use S1Seq mapping to map the distribution of meiotic DSBs in spo11 mutant strains of Saccharomyces cerevisiae.
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Project description:Meiotic chromosome architecture called M-bM-^@M-^\axis-loop structuresM-bM-^@M-^] and histone modifications have been demonstrated to regulate the Spo11-dependent formation of DNA double-strand breaks (DSBs) that trigger meiotic recombination. Using genome-wide chromatin immunoprecipitation (ChIP) analyses followed by deep sequencing, we compared the genome-wide distribution of the axis protein Rec8 (the kleisin subunit of meiotic cohesin) with that of oligomeric DNA covalently bound to Spo11, indicative of DSB sites. The frequency of DSB sites is overall constant between Rec8 binding sites. However, DSB cold spots are observed in regions spanning M-BM-10.8 kb around Rec8 binding sites. The axis-associated cold spots are not due to exclusion of Spo11 localization from the axis, since ChIP experiments revealed that substantial Spo11 persists at Rec8 binding sites during DSB formation. Spo11 fused with Gal4 DNA binding domain (Gal4BD-Spo11) tethered in close proximity (M-bM-^IM-$0.8 kb) to Rec8 binding sites hardly forms meiotic DSBs, in contrast with other regions. In addition, H3K4 tri-methylation (H3K4me3) remarkably decreases at Rec8 binding sites. These results suggest that reduced histone H3K4me3 in combination with inactivation of Spo11 activity on the axis discourages DSB hot spot formation. ChIP-seq analyses of Rec8, Spo11, and Gal4BD-Spo11 on budding yeast meiotic chromosomes M-bM-^@M-" Distribution of Rec8 in wt and Gal4BD-Spo11-expressing cells at 4h after meiotic induction M-bM-^@M-" Distribution of Spo11 at 3h, 4h, and 5h after meiotic induction M-bM-^@M-" Distribution of Gal4BD-Spo11 at 0h after meiotic induction
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Project description:DNA double-strand breaks (DSBs) initiate meiotic recombination. Past DSB-mapping studies have used rad50S or sae2? mutants, which are defective in break processing, to accumulate DSBs, and report large (= 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2? mutants. We therefore developed novel methods that detect DSBs using ssDNA enrichment and microarray hybridization, and that use background-based normalization to allow cross-comparison between array datasets, to map genome-wide the DSBs that accumulate in processing-capable, repair-defective dmc1î and dmc1î rad51î mutants. DSBs were observed at known hotspots, but also in most previously-identified “DSB-cold” regions, including near centromeres and telomeres. While about 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1? shows that most of these regions have significant DSB activity. Thus, DSBs are distributed much more uniformly than was previously believed. Southern-blot assays of DSBs in selected regions in dmc1?, rad50S and wild-type cells confirm these findings. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as the primary strand transfer activity genome-wide, and Spo11-induced lesions as initiating all meiotic recombination. Keywords: DSB mapping, ChIP-chip, single strand DNA , BND cellulose We use two different strategies to map the genome-wide distribution of meiotic DSBs in the yeast Saccharomyces cerevisiae. The first is a chromatin immunoprecipitation (ChIP) based approach that targets the Spo11p protein, which remains covalently attached to DSB ends in the rad50S mutant background. The second approach involves BND cellulose enrichment of the single strand DNA (ssDNA) recombination intermediate formed by end-resection at DSB sites following Spo11p removal. We use dmc1 and dmc1 rad51 mutants that accumulates meiotic single strand DNA intermediates
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